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Engineering bacterium, and application thereof in production of alpha-ketoglutaric acid

A technology of ketoglutaric acid and glutamic acid, applied in microorganism-based methods, bacteria, microorganisms, etc., can solve problems such as affecting yield and purification effect, and achieve good industrial application prospects, easy availability of raw materials, and simple production process. Effect

Active Publication Date: 2018-12-07
卓虹超源生物科技(郑州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Engineering bacteria containing L-glutamic acid oxidase (CN201410146961.3, CN201210563671.X, etc.) can produce α-ketoglutarate, but some will produce hydrogen peroxide during this process, even if co-expressed hydrogen peroxide Enzymes or external addition of hydrogen peroxidase cannot prevent the oxidation of α-ketoglutarate to produce succinate, which affects the yield and purification effect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Screening of highly active L-glutamic acid dehydrogenase producing bacteria with NAD as coenzyme and its L-glutamic acid dehydrogenase gene. The screening medium formula is: sodium chloride 3g / L, potassium dihydrogen phosphate 1g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 0.2g / L, NAD0.01g / L, L-glutamic acid 1g / L , pH7. The culture temperature is 30°C, and the sterilized medium is used to enrich the fast-growing strains in the soil material. After the primary screening and secondary screening, a strain that can use glutamic acid to grow rapidly is finally obtained. According to the conservation of L-glutamate dehydrogenase, degenerate primers were designed, and a gene was cloned, named gdhCN, as shown in the sequence table SEQ ID NO:32. And after being connected to the first T7 promoter of the pETDuet-1 plasmid, it was transformed into Escherichia coli BL21(DE3) to obtain the Escherichia coli BL21(DE3) / pETDuet-1-gdhCN strain, and the expression was determ...

Embodiment 2

[0054] Recombinant Escherichia coli construction: the gene encoding NADH oxidase was respectively connected to the second T7 promoter of the plasmid pETDuet-1-gdhCN. Various double-gene co-expression recombinant plasmids were obtained, the plasmids were transformed into Escherichia coli BL21 (DE3), and positive transformants were obtained by screening with ampicillin plate, that is, recombinant Escherichia coli was obtained.

[0055] After the expression was induced according to the method described in Example 1, the collected cells were subjected to transformation analysis, and the results are shown in Table 2. The whole cell transformation system in the transformation system is: cell wet weight 100g / L, L-glutamic acid 50g / L, pH 8.0, temperature 35°C, shaker speed 250 rpm; transformation time 1 hour.

[0056] Table 1 Comparison of the efficiency of producing α-ketoglutarate in Escherichia coli co-expressed with double genes of L-glutamate dease and NADH oxidase

[0057] ...

Embodiment 3

[0060] According to the strain construction method described in Example 1 (various types of plasmids were screened for positive transformants using different resistance plates according to the instructions) and the induced expression method, various types of cells were collected for transformation analysis, and the results are shown in Table 2. The whole cell transformation system in the transformation system is: cell wet weight 20g / L, L-glutamic acid 50g / L, pH 9.0, temperature 30°C, shaker speed 250 rpm; transformation time 10 hours.

[0061] Table 2 The comparison of various expression plasmids for the transformation to produce α-ketoglutarate

[0062] strain

[0063] It can be seen from the above table that the effect is the best when pETDuet-1 is used for co-expression.

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Abstract

The invention discloses an engineering bacterium, and an application thereof in the production of alpha-ketoglutaric acid, and belongs to the technical field of bioengineering. The engineering bacterium is a recombinant strain capable of producing pure alpha-ketoglutaric acid at low cost, simultaneously expresses exogenous L-glutamate dehydrogenase and NADH oxidase, and is obtained by knocking outan alpha-ketoglutaric acid absorption gene from host Escherichia coli; and the engineering bacterium can achieve enhanced expression of any one or more of a lactic acid transporter gene, an alpha-ketoglutaric acid transporter gene and an NAD synthesis gene. A method for producing the alpha-ketoglutaric acid by using the recombinant bacterium has the advantages of simple process, few impurities and great industrial application values.

Description

technical field [0001] The invention relates to an engineering bacterium and its application in producing α-ketoglutarate, belonging to the technical field of bioengineering. Background technique [0002] α-Ketoglutaric acid (α-Ketoglutaric acid) is a weakly acidic organic acid. α-Ketoglutarate is widely used in chemical industry, food, medicine and other fields. [0003] At present, the production of α-ketoglutaric acid mainly includes chemical and biological methods. The production of α-ketoglutaric acid by chemical method has high cost and serious pollution. Biological methods mainly include L-glutamic acid conversion method and direct fermentation method. Fermentation of α-ketoglutarate using glucose as raw material (CN201210056106.4, CN201110329273.7, etc.) usually takes a long time to ferment, and the cost of extracting α-ketoglutarate from the impurity fermentation broth system is relatively high. Engineering bacteria containing L-glutamic acid oxidase (CN20141014...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/50C12R1/19
CPCC07K14/245C12N9/0016C12N9/0036C12P7/50C12Y104/01002C12Y106/99003
Inventor 蔡宇杰熊天真蒋静丁彦蕊白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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